In order to examine the potential of HSV-1 as a vector to study the expression of DNA repair genes in mammalian cells, a recombinant virus containing the den V gene from bacteriophage T4 has been constructed. This gene encodes a pyrimidine dimer-specific endonuclease that has the capacity to initiate excision repair of DNA. Transfection studies indicate that excision repair deficient xeroderma pigmentosum (XP) group A cells are able to carry out excision repair initiated by the den V gene product. This gene along with the 3' LTR of Rous Sarcoma Virus and the SV40 polyadenylation signals were inserted into the non-essential glycoprotein I gene of HSV-1. Immunoprecipitation studies confirmed the production of the den V protein in virus infected cells. The uv survival of this HSV-1:den V recombinant virus was examined in various primary cell types. The cells examined in this study were primary fibroblasts from a normal individual, a Trichothiodystrophy patient and five XP patients as well as a mouse L cell line. The ability of the virally encoded den V gene to restore the excision repair deficiency in these cells was measured by monitoring the uv survival of HSV-1:den V as compared to wildtype HSV-1. Increased survival of HSV-1:den V was detected in Trichothiodystrophy cells, and in cells from XP complementation groups A, C and D, but not in XP cells from complementation groups E and F or in mouse L cells. These results demonstrate that HSV can be effectively used to study the expression of a cloned DNA repair gene in a variety of cell types. HSV has a substantial capacity of gene insertion and a wide host range including cells of human and rodent origin. / Thesis / Master of Science (MS)
| Identifer | oai:union.ndltd.org:mcmaster.ca/oai:macsphere.mcmaster.ca:11375/23096 |
| Date | 09 1900 |
| Creators | Tang, Katherine |
| Contributors | Rainbow, Andrew, Biology |
| Source Sets | McMaster University |
| Language | English |
| Detected Language | English |
| Type | Thesis |
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